1. Field of the Invention
The present invention is broadly concerned with a method and apparatus designed to greatly facilitate addition of fluids to, or removal of fluids from, an internal body organ or part of an animal. More particularly, it is concerned with such a method and apparatus which is especially suited for the large scale production of monoclonal antibodies in large mammals, and which permits repeated monitoring and/or nutritional enhancement of the in vivo monoclonal antibody production procedure. In preferred methods, monoclonal antibody production is enhanced by in vitro education of the cells using fluid characteristic of the host organ; moreover, use of the allantois of a pregnant host (e.g., cow, horse, sheep or pig) after the onset of fetus immunocompetency is especially advantageous.
2. Description of the Prior Art
When a foreign substance enters the body of a vertebrate animal or is injected into it, one aspect of the immune response is the secretion by plasma cells of antibodies. Quite apart from the natural function of antibodies in the animal's immune response, such antibodies have long been an important tool for investigators, who capitalize on their specificity to identify or label particular molecules or cells and to separate them from a mixture. The antibody response to a typical antigen is normally highly heterogeneous, and even the best of antisera are really heterogeneous mixtures of many different antibody molecules that vary in charge, size, and in such biologic properties as the ability to fix complement or to agglutinate or precipitate antigen. It is extremely difficult to separate the various antibodies in antisera, and therefore conventional antisera contain mixtures of antibodies, and such mixtures vary from animal to animal.
It is also known that malignant tumors of the immune system (called myelomas) are characterized by rapidly proliferating cells producing large amount of abnormal immunoglobulines called myeloma proteins. A tumor itself is considered to be an immortal clone of cells descended from a single progenitor, and so myeloma cells can be cultured indefinitely, and all the immunoglobulins they secrete are identical in chemical structure. They are in effect monoclonal antibodies, but there is no way to know what antigen they are directed against, nor can one induce myelomas that produce antibody to a specific antigen. However, in recent years researchers have learned how to fuse myeloma cells of mice with lymphocytes from the spleen of mice immunized with a particular antigen. The resulting hybrid-myeloma, or "hybridoma" cells express both the lymphocyte's property of specific antibody production and the immortal character of the myeloma cells. Such hybrid cells can be manipulated by the techniques applicable to animal cells in permanent culture. Individual hybrid cells can be cloned, and each clone produces large amounts of identical antibody to the single antigenic determinant. The individual clones can be maintained indefinitely, and at any time samples can be grown in culture or injected into animals for large scale production of monoclonal antibody. Highly specific monoclonal antibodies produced by this general method have proved to be a versatile tool in many areas of biological research and clinical medicine.
While the utility of specific monoclonal antibodies is manifest, a problem has arisen because of the difficulty of producing significant (e.g., liter) quantities of the antibodies. Obviously, the production of such antibodies in mice is not at all suited for large scale production.
In response to this problem, it has been suggested to employ large mammals such as cattle or sheep for in vivo production of monoclonal antibodies. In one such procedure, the cells of hybridoma clones are introduced into the amniotic fluid of a cow in the early stages of gestation prior to the onset of fetal immunocompetency and are allowed to multiply. After a suitable growth period, quantities of monoclonal antibodies can be harvested. While the above described technique shows considerable promise, a number of practical problems remain. For example, it is desirable to monitor the production of monoclonal antibodies during incubation thereof, and the problems of obtaining samples of the amniotic fluid on a frequent recurrent basis are formidable. The straightforward procedure of simply making a laparotomy incision in the cow's body, manipulation of the uterus, introducing or withdrawing materials, can create multiple insults to the cow, uterus and fetus, which may lead to premature death, infection, or abortion of the fetus. By the same token, in order to enhance antibody production in the amniotic fluid, it is oftentimes desirable to introduce nutrient fluids into the uterus. Here again, the conventional techniques for such introduction, if used repeatedly, can cause severe problems to the animal and uterine environment.
Prior art patents describing various types of percutaneous transport tubes and related structure include: U.S. Pat. Nos. 4,315,513, 3,401,689, 3,515,124, 3,570,484, 3,583,387, 3,961,632 and 3,333,588. All of the structures described in the aforementioned patents are deficient in important respects, and would present serious problems if it were attempted to use the same in the context of in vivo monoclonal antibody production.
In addition, the prior practice of simply placing a cell line in amniotic fluid often leads to rapid death of a large proportion of cells. That is to say, cell lines of interest are typically cultured in highly specific and optimized media, and under relative critical conditions. For example, many cell lines are cultured and allowed to multiply at incubation temperatures of 37 degrees centigrade in specially prepared media, with periodic screening and feeding at regular intervals (e.g., every 48-72 hours). Prior to inoculation, the cells are concentrated into a fixed volume of their media plus fetal calf serum and are surgically implanted. However, this procedure gives only mediocre results, and can often fail completely in that the cells do not multiply or do not produce the desired antibody. One problem with this approach is that the cells, when inoculated, can experience severe "shock" owing to the radically different ambient environment of the amniotic fluid as compared with the previous in vitro culture media and scrupulously maintained growth conditions.
Finally, use of amnionic fluid of a pregnant host as a growth media for hybridoma cells leads to a number of additional difficulties. Specifically, in the case of cattle, it is known during the second trimester of gestation the amnionic fluid consistency changes and it becomes gel-like. Moreover, the fetus swallows substantial quantities of the amniotic fluid, especially during the first trimester of gestation. For the foregoing reasons, prior in vivo attempts to produce monoclonal antibodies using amnionic fluid have been conducted during early stages of gestation so that the entire procedure is accomplished prior to the onset of fetal immunocompetency. If the procedure is carried on after the fetus becomes immunocompetent, the fetus will treat the inoculated cells as antigens and will develop appropriate antibodies, thereby killing the cells. Accordingly, the gestational time frame for cell growth is severely limited and critical in the case of amnionic fluid. Also, surgical manipulation of the fetus and amnionic sac during the early stages of pregnancy is difficult (because of uterine anatomical positioning, flank incisions, high paralumbar fossa, must be utilized), and this can lead to spontaneous abortion and/or fetal infection. Thus, while use of the amnionic fluid of a pregnant host is theoretically possible, a number of practical problems remain.
It will therefore be seen that there is a real and unsatisfied need in the art for methods and apparatus which permit easy, rapid addition to fluids to, or removal of fluids from, specific body parts or organs of animals, so as to facilitate the production and monitoring of monoclonal antibodies, while at the same time avoiding repeated insults to the animal and other internal organs and problems of unintended cell death and low levels of antibody production.